The invention relates generally to the field of laser capture microdissection (LCM). More particularly, the invention relates to an automated system for performing LCM. Specifically, preferred embodiments of the invention relate to an automated LCM system that includes a automated single cell and tissue area targeting capability based on fluorescence labeling and image analysis, an automated cap arm mechanism, a road map camera, and a virtual joystick.
Laser capture microdissection (LCM) is a rapid, reliable method for procuring pure populations of targeted cells from specific microscopic regions of tissue sections for subsequent analysis. LCM-based molecular analysis of histopathological lesions can be applied to any disease process that is accessible through tissue sampling. Examples include mapping the field of genetic changes associated with the progression of microscopic premalignant cancer lesions; analysis of gene expression patterns in multiple sclerosis, atherosclerosis and Alzheimer""s disease plaques; infectious microorganism diagnosis; typing of cells within disease foci; and analysis of genetic abnormalities in utero from selected rare fetal cells in maternal fluids.
The LCM technique is generally described by Emmert-Buck et al., Science 274, 998(1996), the entire contents of which are incorporated herein by reference. The purpose of the LCM technique is to provide a simple method for the procurement of selected human cells from a heterogeneous population contained on a typical histopathology biopsy slide. In an LCM method, a transfer surface is placed onto the tissue section and then focally bonded to the targeted tissue, allowing it to be selectively removed for molecular analysis. In the microscope, the operator views the tissue and selects microscopic clusters of cells for analysis, then activates a laser within the microscope optics. The pulsed laser beam is absorbed within a precise spot on the transfer film immediately above the targeted cells. At this precise location, the film melts and fuses with the underlying cells of choice. When the film is removed, the chosen cells remain bound to the film, while the rest of the tissue is left behind.
LCM offers the advantage of transferring cells of interest to the polymer film in one step. The separate fragmentation step used in conventional microdissection and the resulting contaminating debris are avoided. In addition, only the targeted cells are affected such that the remaining tissue on the slide is fully accessible for further capture, allowing comparative molecular analysis of adjacent cells. The exact morphology of the procured cells is retained and held on the transfer film. In contrast to manual microdissection where cells may be pulverized or lost the procurement of specific cells from a complex tissue section by LCM is reduced to a routine method amenable to widespread research and clinical diagnostic use.
In a manually operated laser capture microdissection system, the operator looks through a microscope at a tissue biopsy section mounted on a standard glass histopathology slide, which typically contains groups of cells. A thermoplastic film is placed over and in contact with the tissue biopsy section. Upon identifying a cell, or group of cells, of interest within the tissue section, the operator centers them in a target area of the microscope field and then generates a pulse from a laser such as a carbon dioxide laser having an intensity of about 50 milliwatts (mW) and a pulse duration of between about 50 to about 500 milliseconds (mS). The laser pulse causes localized heating of the plastic film as it passes through it, imparting to it an adhesive property. The cells then stick to the localized adhesive area of the plastic tape directly above them, whereupon the cells are extracted and readied for analysis. Because of the small diameter of the laser beam, extremely small clusters of cells may be microdissected from a tissue section.
By taking only these target cells directly from the tissue sample, researchers can immediately analyze the gene and enzyme activity of the target cells using other research tools. Such procedures as polymerase chain reaction amplification of DNA and RNA, and enzyme recovery from the tissue sample have been demonstrated. No limitations have been reported in the ability to amplify DNA or RNA from tumor cells extracted with laser capture microdissection.
A typical tissue biopsy sample consists of a 5 to 10 micron slice of tissue that is placed on a glass microscope slide using techniques well known in the field of pathology. This tissue slice is a cross section of the body organ that is being studied. The tissue consists of different types of cells. Often a pathologist desires to remove only a small portion of the tissue for further analysis.
A method for automating a laser capture microdissection is provided comprising providing a fluorescently-labeled tissue sample on a microscope slide, wherein the fluorescent label on the tissue corresponds to a biological property of interest; providing a source of fluorescent excitation, wherein an excitation beam emitted by the source is of an intensity and wavelength to excite a fluorescent label associated with the labeled tissue sample; exciting the tissue sample with the excitation beam and recording at least one information corresponding to an excitation pattern of the tissue sample; selecting from the recorded information, at least one section of the tissue sample for capture by laser capture microdissection; and targeting a laser for selectively capturing the at least one section of the tissue sample by laser capture microdissection.
In the method the at least one information corresponding to an excitation pattern of the tissue sample is a set of positional coordinates of sections of the tissue sample with increased fluorescence. The source of fluorescent excitation can be an EPI laser lamp. The method may further comprise: analyzing the recaptured image of the fluorescent tissuesample by scanning the image for locations of enchanced fluorescence and responsive to the scanned information, selecting one or more sections of the tissue sample for laser capture microdissection.
In another aspect the method comprises analyzing the captured image of the fluorescent tissue sample by displaying the image in a video monitor; and selecting locations of enhanced fluorescence on the tissue sample by inputting a selection into an I/O device.
One embodiment of the invention provides an automated cap transfer system comprising a horizontal bar coupled to a main support bar by a vertical lead screw, whereby operation of the lead screw actuates a vertical displacement of the horizontal bar relative to the support bar; a fork coupled to the horizontal bar, the fork having two or more arms for engaging a LCM cap; a pivotable weight coupled to the horizontal bar, wherein the weight is seated on an engaged LCM cap; a lever coupled to the horizontal bar, the lever comprising at least one pin for engaging the weight and a pivot axis; a kick bar coupled to the support bar, whereby lowering the horizontal bar causes the kick bar to engage the lever and actuate a pivot of the lever about the pivot axis thereby causing the at least one pin of the lever to engage the weight and displace the weight relative to the cap.
In another aspect an automated method of cap transfer in a LCM is provided comprising providing a work surface comprising a translation stage for performing LCM; providing a cap transfer arm coupled to the work surface for removably engaging a LCM cap; providing a controller coupled to a memory for receiving and storing an information corresponding to one or more locations on the work surface; and operating a movement of the cap transfer arm to place and remove a cap from one or more locations on the work surface.